Cancer Cell Membrane-Coated Adjuvant Nanoparticles with Mannose

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Cancer Cell Membrane-Coated Adjuvant Nanoparticles with Mannose Modification for Effective Anticancer Vaccination Rong Yang, Jun Xu, Ligeng Xu, Xiaoqi Sun, Qian Chen, Yuhuan Zhao, Rui Peng, and Zhuang Liu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b09041 • Publication Date (Web): 17 May 2018 Downloaded from http://pubs.acs.org on May 17, 2018

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Cancer Cell Membrane-Coated Adjuvant Nanoparticles with Mannose Modification for Effective Anticancer Vaccination

Rong Yang, Jun Xu, Ligeng Xu*, Xiaoqi Sun, Qian Chen, Yuhuan Zhao, Rui Peng*, Zhuang Liu*

Institute of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-based Functional Materials and Devices, Soochow University, Suzhou, Jiangsu, 215123, China E-mail: [email protected], [email protected], [email protected]

ABSTRACT Tumor vaccines for cancer prevention and treatment have attracted tremendous interests in the area of cancer immunotherapy in recent years. In this work, we present a strategy to construct cancer vaccines by encapsulating immune-adjuvant nanoparticles with cancer cell membrane modified by mannose. Poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles are firstly loaded with toll-like receptor 7 agonist, imiquimod (R837). Those adjuvant nanoparticles (NP-R) are then coated with cancer cell membranes (NP-R@M), whose surface proteins could act as tumor-specific antigens. With further modification with mannose moiety (NP-R@M-M), the obtained nanovaccine shows enhanced uptake by antigen presenting cells such as dendritic cells (DCs), which would then be stimulated to the maturation status to trigger antitumor immune responses. With great efficacy to delay tumor development as a prevention vaccine, vaccination with such NP-R@M-M in 1

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combination with checkpoint-blockade therapy further demonstrates outstanding therapeutic efficacy to treat established tumors. Therefore, our work presents an innovative way to fabricate cancer nanovaccines, which in principle may be applied for a wide range of tumor types.

KEYWORDS: vaccination, cancer cell membrane, cancer immunotherapy, checkpoint blockade, mannose modification

By utilizing the patients' own immune system instead of cytotoxic agents (e.g. chemotherapy) to recognize and destruct cancer cells, cancer immunotherapy often shows high specificities and low toxicities to eradicate tumors and prevent their recurrence.1-6 The current cancer immunotherapies, such as cancer vaccines,7-10 immune checkpoint blockade immunotherapy,11-12 adoptive cell therapy (e.g. CAR-T),13 have shown encouraging clinical results to treat different types of tumors. Among various classes of cancer immunotherapy strategies, cancer vaccines are featured with relatively low cost and high specificity to attack tumor cells with low side effects.14-17 An effective nanovaccine is often composed of the antigen, adjuvant, and possibly the delivery carrier.18-19 For typical tumor vaccines, tumor-specific peptides, proteins, DNA or even mRNA could be utilized as the antigen to trigger specific antitumor immunities. Although highly specific, those tumor vaccines may show significantly varied efficacies towards different cancer patients, whose tumor antigen expression profiles may have large differences between individuals.3,

20

Another

strategy to develop tumor vaccines that may be applied for different tumor types is to use whole tumor cell lysates with mixed tumor-associated antigens.21 However, the majority of tumor lysate contents, especially those intracellular components, may not be effective in triggering antitumor 2


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immune responses. Considering the fact that in cancer immunotherapy, cytotoxic T lymphocytes (CTLs) usually recognize tumor cells via binding with receptors on the cancer cell membrane,22 it would be thus interesting to develop cancer vaccines using tumor cell membranes as the tumor-specific antigen. The technology of encapsulating nanoparticles (NPs) with membranes of different cell types has been proposed to be an interesting approach in biomedical engineering for various purposes.14, 23-25 As firstly reported by Zhang and co-workers, NPs coated with bacteria membrane could act as effective antibacterial vaccines.14 Moreover, NPs coated with red blood cell membrane and further loaded with both antigen peptides and adjuvant have also been proposed to be an alternative strategy to construct nanovaccine against cancer.23, 26-27 However, the construction of nanovaccines based on cancer cell membrane-coated NPs has not yet been reported until a very recent report by the Zhang group.28 Nevertheless, there is a lack of specific targeting to antigen presenting cells (APCs) for the cell-membrane-coated nano-vaccine designed in that work. Herein, we construct a nanovaccine formulation by coating adjuvant nanoparticles with mannose-modified tumor cell membranes for cancer immunotherapy. In our design, Poly (D, L-lactide-co-glycolide) (PLGA) NPs are loaded with R837, an agonist against toll-like receptor 7 (TLR-7),29-30 and then encapsulated with membranes from B16-OVA cancer cells. The obtained NP-R@M is further modified with mannose by a lipid-anchoring method. Owing to the specific binding between mannose and its receptors on APCs such as dendritic cells (DCs),16,

31

our NP-R@M-M shows enhanced DC uptake and stronger

stimulation effect to trigger DC maturation. At the in vivo level, NP-R@M-M upon intradermal injection could effectively migrate to draining lymph nodes (LNs) and trigger tumor-specific immune responses.32-33 Such NP-R@M-M on itself could act as an effective prophylactic vaccine to 3


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delay the tumor development, and if in combination with anti-programmed death-1 (anti-PD-1) checkpoint blockade could further offer great therapeutic effect to treat established B16-OVA melanoma tumors. Our work highlights the great potential of membrane-coated NPs as an attractive tool to develop nanovaccines for cancer immunotherapy.

RESULTS AND DISCUSSION

The fabrication process of NP-R@M-M is illustrated in Figure 1&Figure 2a. PLGA NPs with or without R837 loading were prepared following a well-established protocol as described earlier.22 The R837 loading capacity in the obtained NP-R nanoparticles was determined to be 1 : 100 (R837 : PLGA, w/w) by a high performance liquid chromatography (HPLC, Agilent 1260). The loading of R837 within PLGA nanoparticles was found to be rather stable (Supporting Information, Figure S1). Cancer cell membranes were collected by freezing and thawing cancer cells following the literature protocol.34 In order to coat the PLGA cores with cancer cell membranes, we simply mixed the NP or NP-R sample with purified cell membranes at 4oC overnight. Then, the obtained NP@M or NP-R@M nanoparticles were collected by centrifugation at 10000 rpm for 10 min. To further modify NP@M or NP-R@M nanoparticles with mannose, the obtained membrane-coated NPs were mixed with

mannose-conjugated

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-

N-methoxy

(polyethylene glycol) (DSPE-PEG-Man), whose long carbon-hydrogen chains would be automatically inserted into the lipid bilayer structure of cell membrane. We then carefully characterized the obtained PLGA NPs and NP@M nanoparticles. As revealed by transmission electron microscopy (TEM), a clear shell, which should be the cell membrane layer, was observed on the surface of membrane-coated NPs, but not on bare PLGA NPs (Figure 2b, 4


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Supporting Information, Figure S2).

14, 16, 35-38

As illustrated by the bicinchoninic acid (BCA)

protein content assay, while the bare NP showed undetectable protein content, NP@M and NP-R@M samples showed significant and similar amounts of protein contents (Figure 2c). Upon coating with the cell membrane, the resulting NP@M showed slightly increased sizes to be ~120 nm in diameter, while the final NP-R@M and NP-R@M-M were approximately 140 and 160 nm in size (Figure 2d), respectively. Note that the nanoparticle size became larger after mannose modification, likely owing to the additional PEG chains anchored on the surface of nanoparticles after NP-R@M was modified by DSPE-PEG-Man. After membrane coating, the surface zeta potential of nanoparticles slightly decreased to be −23 mV. (Supporting Information, Figure S3). Given the crucial roles of DCs in regulating immune responses, the interactions between the nanovaccine and DCs would greatly determine the quality of induced immunities.13 Firstly, the potential cytotoxicity of NP@M-M, NP-R@M and NP-R@M-M towards bone marrow-derived DCs (BMDCs) was determined by the cell viability assay. The negligible cytotoxicity of NP@M, NP@M-M, and NP-R@M-M toward BMDCs implied the high compatibility of our antigen delivery system (Supporting Information, Figure S4). It is important for immune responses that tumor-associated antigens are processed and presented by APCs. Therefore, the cellular uptake profile of antigen-loaded NPs by APCs would be important for the efficacy of nanovaccines. PLGA cores were loaded with the lipophilic fluorescence dye 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine Perchlorate (DiD) to facilitate in vitro tracking, obtaining DiD-NP@M and DiD-NP@M-M nanoparticles. To evaluate the cell uptake of NP@M-M by APCs, BMDCs were incubated with DiD-NP@M or DiD-NP@M-M nanoparticles for the flow cytometric analysis (Figure 3a&Supporting Figure S5). As shown in Figure 3b, 5


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DiD-NP@M-M showed significantly enhanced cellular uptake by BMDCs compared to DiD-NP@M without mannose modification. Such a phenomenon was also confirmed by confocal fluorescence imaging of BMDCs incubated with the two types of NPs (Figure 3c). Similarly, owing to the over expression of mannose receptors on macrophages, we also found that mannose modified DiD-NP@M-M showed more efficient cellular uptake by macrophages compared to DiD-NP@M (Supporting Information, Figure S6a-b). The enhanced cell uptake of antigen / adjuvant co-loaded NPs by APCs would be greatly favorable for inducing stronger immune responses and more effective vaccination. The immature DCs after capturing antigens could be stimulated into the matured status, leading to antigen presentation on their surface to further activate T cells and induce the subsequent immune responses.

39-40

Afterwards, the maturation of DCs would occur, accompanying with Therefore,

BMDCs were incubated with different formulations of NPs, with lipopolysaccharides (LPS) used as the positive control. The abilities of those nanoparticles to stimulate DC maturation were evaluated by flow cytometry analysis, using CD80 and CD86 as the surface markers for mature DCs (Figure 4a). It was found that while bare PLGA NPs showed no appreciable DC stimulation effect, NP-R with loading of R837 a TLR-7 agonist as the adjuvant, NP-M with coating of tumor cell membranes, as well as NP-R@M with both R837 loading and cell membrane coating, all triggered significant DC maturation, with NP-R@M found to be stronger than the other two. Moreover, the DC stimulation effect could be further improved by mannose modification. Notably, the NP-R@M-M formulation appeared to be highly effective in triggering DC maturation, to a level close to that achieved by LPS (Figure 4a & Figure 4b). Meanwhile, to evaluate DC maturation, we also tested the secretion of cytokines by BMDCs.41 6


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Supernatants recovered from DCs after 12 h of incubation with various NP vaccines were tested by enzyme linked immunosorbent assay (ELISA), to determine secretion of tumor necrosis factor-α (TNF-α) (Figure 4c) and interleukin-12 (IL-12p40) (Figure 4d), which are cytokines highly relevant to antitumor immunity.

42-44

Compared to samples treated with free NPs, those incubated with other

NP formulations including NP@M, NP-R, NP-R@M, NP@M-M and NP-R@M-M showed significantly increased secretion levels of TNF-α and IL-12p40. Moreover, the highest levels of TNF-α and IL-12p40 secretion were found from DCs after treatment with NP-R@M-M, consistent to the DC maturation data. Therefore, those data taken together indicate that our nanovaccine formulation, NP-R@M-M, with R837 loading as the adjuvant, membrane coating to provide tumor-specific antigens, and mannose modification to enhance APC uptake, appears to be the most effective for in vitro activation of DCs. Generally speaking, whether antigen and adjuvant can efficiently migrate into lymph nodes and interact with APCs would greatly determine the quality of induced immune responses.45-46 Therefore, we firstly evaluated the in vivo migration efficacy of the nanovaccine labeled with DiD using fluorescence imaging. Mice were injected with DiD-NP-R@M or DiD-NP-R@M-M at left footpad and then the migration was monitored at 1 h, 3 h and 6 h post injection by in vivo fluorescence imaging (Figure 5a&5b). Ex vivo fluorescence imaging of lymph nodes taken at 6 h post injection of nanoparticles further confirmed that the lymph node retention of DiD-NP-R@M-M was much higher than that of DiD-NP-R@M (Figure 5c, d). Therefore, our mannose modified vaccine nanoparticles showed obviously enhanced retention within lymph nodes after local administration, likely owing to their more efficiently interaction with APCs (mannose receptor positive) within lymph nodes, favorable for inducing robust in vivo immune responses. It is known that activated DCs will enter 7


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lymph vessels and accumulate in the T cell zone in the draining lymph nodes to interact with native T cells.47 As expected, accumulation of DCs within the T cell zone of the draining popliteal lymph nodes was observed based on immunofluorescence imaging data (Supporting Figure S7). Next, we studied the in vivo prophylactic effects of the nanovaccine using B16-OVA melanoma tumor model. As depicted in Figure 6a, mice were intradermally immunized with different vaccine formulations including NP@M, NP@M-M, NP-R@M, and NP-R@M-M for three times at one week interval and then challenged by B16-OVA melanoma cells at day 7 post the final immunization. The tumor growth was monitored starting from day 9 post tumor cells inoculation. It was found that melanoma cell membrane-coated NPs (NP@M) could partially inhibit tumor progression compared to untreated (PBS) group (Figure 6b). Treatment by NP-R could also slightly delay the tumor growth, likely owing to the non-specific immune stimulation effect by those adjuvant nanoparticles. When TLR7 agonist (R837) was integrated into the nanovaccine formulation, the obtained NP-R@M nanovaccine showed increased tumor growth inhibition efficacy compared to the NP@M vaccine formulation (Figure 6b). Furthermore, when modified with mannose, the obtained NP-R@M-M nanovaccine exhibited the strongest antitumor efficacy compared to all other formulations, including NP-R@M (Figure 6b). However, when those immunized mice were challenged with 4T1 mouse breast tumor cells, NP-R@M-M did not show any antitumor efficacy (Figure 6c), evidencing the specificity of antitumor immunities induced by our developed nanovaccine. All these results greatly support the feasibility and validity of our design on nanovaccine involving the cancer cell membrane as the tumor-specific antigen, TLR agonist as the adjuvant, and mannose as the APC recognition moiety to further promote vaccination efficacy. To uncover the underlying mechanisms of the developed nanovaccine in combating tumor 8


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progression, immunized mice were sacrificed at day 7 post the final immunization. Considering the critical roles of CTLs-mediated immune response in tumor treatment, the generation of CD107a, the functional marker of CTLs degranulation,48 was evaluated using flow cytometry assay. It was found that different formulations of cancer cell membrane coated NPs could all induce increased generation of CD3+CD8+CD107a+ T cells in the spleen compared with the untreated group (Figure 6d), particularly for those with R837 loading. As another typical marker of the cytotoxic activity of CTLs, the production level of interferon gamma (IFN-γ) in sera of immunized mice was also determined using ELISA. Interestingly, it was uncovered that only NP-R@M-M nanovaccine formulation could trigger the up-regulation of IFN-γ generation (Figure 6e). All these above properties endowed NP-R@M-M nanovaccine with high efficiency in combating melanoma progression. Recently, checkpoint blockade therapy such as using antibodies against cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1) as well as its ligand

(PD-L1), to relieve immunosuppression milieu and prevent T cells exhaustion, have shown

tremendous promises in the clinic when used alone or in combination with other types of therapies.49-51 Due to its properties in inducing specific antitumor responses, cancer vaccine may be combined with checkpoint blockade therapy to achieved further enhanced therapeutic outcomes, as demonstrated by in both preclinical15 and clinical studies52 in recent years. Therefore, as depicted in Figure 7a, we subsequently explored the potential of our NP-R@M-M nanovaccine to treat established B16-OVA tumors in combination with anti-PD-1 blockade strategy. In our experiments, mice bearing B16-OVA tumors were intradermally immunized with NP-R@M-M for three times on day 4, day 11 and day 18. Anti-PD-1 (dose = 20 µg per mouse) was intravenously injected twice after each round of immunization. It was shown that our developed 9


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NP-R@M-M nanovaccine could effectively inhibit tumor growth at the early stage, similar to that achieved by anti-PD-1 treatment (Figure 7b). However, tumors in these two groups with NP-R@M-M treatment alone or anti-PD-1 treatment alone showed rapid growth later on, resulting in death of all mice within 25-27 days post tumor cells inoculation (Figure 7c). Intriguingly, for mice immunized with NP-R@M-M nanovaccine and treated with anti-PD-1, their tumor progression was effectively inhibited (Figure 7b). Notably, a half of mice (3 out of 6 mice) became tumor free after such combination treatment and survived at day 45 post tumor cells challenge (Figure 7c). Those results strongly evidenced that our developed cancer cell membrane-coated nanovaccine in combination with checkpoint blockade therapy could be a rather attractive strategy for combination cancer immunotherapy.

CONCLUSION In this study, we have successfully constructed a nanovaccine formulation (NP-R@M-M) composed of several key elements involving melanoma cell membrane as the tumor-specific antigen, TLR agonist as the adjuvant, and mannose as an APC-recognition moiety. In such a nanovaccine system, coating of cancer cell membrane may be a general strategy to develop personalized tumor-specific vaccines against different types of tumors. Loading of R837 as a robust adjuvant could greatly promote the immunogenicity of cancer cell membrane coated NPs. Moreover, mannose modification could facilitate the binding and cellular uptake of vaccine NPs by APCs, and further enhance the lymph node retention of nanovaccine for more effective in vivo vaccination. Therefore, our developed nanovaccine, NP-R@M-M, could not only be utilized as prophylactic vaccine to protect mice from tumor cells challenge, but also as therapeutic vaccine to effectively combat 10


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melanoma progression when combining with anti-PD-1 checkpoint blockade therapy. Importantly, such membrane-coated nanovaccine shows great clinical translation potential since all of the materials used in this system are fully biocompatible.

EXPERIMENTAL SECTION Cell line and animals. Murine melanoma cell line B16-OVA (syngeneic with C57BL/6) was grown in the recommended cell culture medium under the standard condition. Female BALB/c mice (6−8 weeks) were maintained under protocols approved by Soochow University Laboratory Animal Center. BMDCs collected from marrow cavities of femurs and tibias of 8- to 10-week-old C57BL/6 mice were cultured in culture dishes containing 4 mL serum-containing RPMI 1640 medium containing 100 mg/mL streptomycin, 10 ng/mL GM-CSF , 100 IU/mL penicillin, 5 ng/mL βmercaptoethanol.53

Synthesis of PLGA-NPs. PLGA-NPs were prepared by the oil-in-water emulsion.54 The PLGA polymer dissolved in dichloromethane (10 mg/mL) was added drop-wisely into 10 mL deionized water, and then stirred overnight to allow the evaporation of dichloromethane solvent. After centrifugation at 4,500 rpm for 20 min to discard the large particles, the PLGA NPs were obtained by centrifugation at 12000 rpm for 30 min and then re-dispersed in distilled water for future use.

Preparation of NP-R@M-M. The B16-OVA-membrane was prepared using liquid nitrogen freezing for 6 times as previously reported.55 Then, the membranes were obtained by centrifugation at 14800 rpm for 10 min. Hence, 11


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the cell nuclei and cytoplasm were removed. The resulting packed B16-OVA membrane were washed once in cold 1 × PBS. Afterwards, B16-OVA membrane was resuspended in 1 × PBS and mixed with PLGA NPs at 4oC overnight. To form mannose-inserted B16-OVA membrane, the solution was stirred for 1 h with DSPE-PEG-Man (0.1 mg/mL), which DSPE-PEG-Man was thus inserted into B16-OVA cell membranes following a previous protocol.29 Then, the mixture was saved in 1 × PBS solution.

Characterization of different NP formulations. The hydrodynamic diameters and zeta potentials of nanovaccine suspended in 1 × PBS were measured by dynamic light scattering (DLS) (Malvern Instruments, UK). The morphology of NP@M was characterized by transmission electron microscope (TEM, JEM-1230, Japan). TEM imaging of those membrane-coated nanoparticles was carried out directly without using any staining. The protein contents in membrane coated NPs were determined by the BCA assay.

Cellular uptake and cytotoxicity studies. In the cell uptake study, the nanoparticles (10 µg) were incubated with 5 × 105 DC cells for 12 h. The DC cells were obtained by centrifugation at 1200 rpm for 3 min. The cell uptake of fluorescently labeled NPs was sorted by flow cytometry (BD FACSCalibur) and confocal microscopy (Becton Dickinson,

San

Jose,

CA).

The

cell

cytotoxicity

was

determined

by

the

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl -2H-tetrazolium bromide (MTT) assay (Sigma) following the standard protocol.

In vivo fluorescence imaging. C57BL/6 mice were intradermally injected at the left footpad with DiD-NP-R@M or 12


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DiD-NP-R@M-M. Fluorescence images were acquired at three different time points post injection using an Lumina Ⅲ (USA) system. The fluorescence images were analyzed by Living Imaging software.

In vitro DC activation and Cytokine Analysis. DCs (5 × 105 cells) were incubated with nanoparticles for 12 h. Flow cytometry analysis was performed to analyze the percentage of mature DCs. Cells suspended in PBS containing 1% FBS were incubated with anti-mouse antibody against CD11c-FITC, CD80-APC, CD86-PE for 20 min at room temperature in dark, and then evaluated by a flow cytometer. For quantitative release of TNF-α and IL-12p40, the DC medium supernatants were measured by ELISA following the vendor’s protocol (eBiosciences).

Tumor challenge and immune response assay. C57BL/6 mice were randomly divided into different groups and vaccinated three times (one week of interval) by intradermal injection of 200 µL (10 mg/mL PLGA, 0.1 mg/mL R837) of different vaccine formulations. Seven days later after the last vaccination, 3.5 × 105 B16-OVA cells were subcutaneously injected into the right flank of each mouse. The tumor sizes were measured every other day according to the following formula = width2 × length × 0.5. Mice were euthanized when the tumor size reached 1500 mm3. For immune response assays, seven days later after the last vaccination, all the mice were sacrificed and the production of IFN-γ in serum was tested by ELISA assay (eBiosciences). To determine the cytotoxicity activity of activated T-cells, splenocytes were stimulated with OVA257-264 and hgp100(25-33) peptides for 4-6 h in the presence of brefeldin A (BFA) 13


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and monensin. Then, the cytoxicity activity was evaluated by staining cells with fluorescence-labeled antibodies against CD3, CD8 and CD107a for flow cytometry analysis following the standard protocol.56

The therapeutic effect of nanovaccine combining with anti-PD-1 blockade immunotherapy. B16-OVA cells (3.5 × 105) were injected subcutaneously at the right flank of each mouse. All the tumor-bearing mice were randomly divided into four groups (six mice per group) for different treatments: PBS (1), NP-R@M-M (2), anti-PD-1 (3), NP-R@M-M + anti-PD-1 (4). On day 4, 11, and 18 after tumor cell implantation, mice were vaccinated with NP-R@M-M by intradermal injection for group 2 and group 4. For mice in group 3 and group 4, mice were i.v. administrated with anti-PD-1 (20 µg per mouse for each injection) on day 5, 8, 12, 15, 19, 22.

ACKNOWLEDGMENTS This work was supported by the National Research Programs from Ministry of Science and Technology (MOST) of China (2016YFA0201200), the National Natural Science Foundation of China (51525203, 51761145041), Collaborative Innovation Center of Suzhou Nano Science and Technology, the ‘111’ program of the Ministry of Education (MOE) of China, and a Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

The Supporting Information is available free of charge on the ACS Publications website: Loading and releasing profiles of R837 from nanoparticles, the TEM image of PLGA NPs, zeta potentials of different types of NPs, cytotoxicity data of membrane coated NPs, flow cytometry data for cells 14


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incubated with fluorescently labeled NP@M or NP@M-M, and immunofluorescence images of the lymph nodes.

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Figure 1. Schematic illustration to show the structure of tumor cell membrane coated, R873 loaded, and mannose modified PLGA NPs (NP-R@M-M), and their functions to induce antitumor immunity as a nanovaccine.

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Figure 2. Preparation and characterization of nanovaccine. (a) Schematic illustration to show the step-by-step preparation of NP-R@M-M nanovaccine. (b) TEM image of NP@M nanoparticles. (c) BCA assay to determine the protein contents for PLGA NPs, NP-R, NP@M, NP-R@M and NP-R@M-M samples. Error bars represent means ± standard deviation (SD) (n = 3). NP and NP-R samples were used as the blank control. The measured ‘protein’ contents in these two samples were due to the absorbance generated from side reactions in the BCA assay. (d) Hydrodynamic sizes of PLGA NPs, NP-R, NP@M, NP-R@M and NP-R@M-M samples measured by dynamic light scattering (DLS).

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Figure 3. Cellular uptake of nanovaccine by BMDCs. (a) Uptake of DiD-loaded NP@M and NP@M-M by BMDCs in vitro. (b) Mean fluorescence intensity of cell uptake capability. (c) CLSM images of cells treated with NP@M and NP@M-M. NP@M and NP@M-M were fabricated with PLGA cores loaded with DiD (red channel) and nucleus was stained with Hoechst (blue channel). Scale bar = 25 µm.

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Figure 4. In vitro DC activation by nanovaccines. (a&b) Representative flow cytometry data (a) and the statistic data (b) to show DC maturation induced by different formulations of NPs. Cells were stained with antibodies against CD11c as the dendritic cell marker, as well as CD80 and CD86 as DC maturation markers (gated from CD11c). (c&d) Secretion of IL-12 p40 (c) and TNF-α (d) from DCs treated with different NP formulations. Error bars represent means ± SD (n = 3). P values were calculated by Student's t test (***P < 0.001, **P < 0.01 or *P < 0.05).

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Figure 5. In vivo and ex vivo fluorescence imaging to show lymph node retention of nanovaccines. (a) Representative in vivo fluorescence images for mice taken at three different time points after intradermal injection of DiD labeled NP-R@M or NP-R@M-M at the hind legs. (b) Quantitative fluorescence signals at the draining lymph nodes based on in vivo imaging data shown in (a). (c) The ex vivo fluorescence image of isolated lymph nodes taken at 6 h after intradermal injection of nanoparticles. (d) Quantification of the fluorescence signals of isolated draining lymph nodes (left) shown in (c). P values were calculated by Student's t test (***P < 0.001, **P < 0.01 or *P < 0.05).

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Figure 6. Antitumor activities of various NP formulations as prophylactic vaccines. (a) Schematic illustration to show the tumor challenge experiment design. (b) The tumor growth curves of B16-OVA tumor grown on mice after pre-treatment with different NP formulations (n ≥ 5, *, P < 0.05). NP-R@M-M showed the strongest prevention effect to delay the tumor progression. (c) The growth curves of 4T1 breast cancer tumors on mice vaccinated with NP-R@M-M prepared with B16-OVA cell membrane. No tumor growth prevention effect of NP-R@M-M was observed for the 4T1 tumor model, demonstrating the specificity of our tumor nanovaccine. (d) The percentages of CD107a positive cells among all T cells measured by flow cytometry after staining of intracellular antigen. (e) IFN-γ production in sera from immunized mice determined by ELISA. P values were calculated by Student's t test (***P < 0.001, **P < 0.01 or *P < 0.05).

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Figure 7. Antitumor therapeutic effect of nanovaccine in combination with anti-PD-1 checkpoint blockade. (a) Schematic illustration to show the experimental design of combining NP-R@M-M vaccination and anti-PD-1 therapy to inhibit tumor growth. (b) Growth curves for B16-OVA tumors on mice after various treatments indicated. (c) Morbidity-free survival of different groups of mice with B16-OVA tumors in (b) after various treatments. P values were calculated by Log-rank (Mantel-Cox) Test (***P < 0.001, **P < 0.01 or *P < 0.05).

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TOC Figure

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Cancer Cell Membrane-Coated Adjuvant Nanoparticles with Mannose

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